Blood is the circulating tissue of an organism that carries oxygen and nutritive materials to the tissues and removes carbon dioxide and various metabolic products for excretion. Whole blood consists of pale yellow or gray yellow fluid, plasma, in which are suspended red blood cells, white blood cells, platelets, and hemostatic factors.
An accurate measurement of the ability of a patient's blood to coagulate and lyse, i.e., hemostasis, in a timely and effective fashion is crucial to certain surgical and medical procedures. Accelerated (rapid) and accurate detection of abnormal hemostasis is also of particular importance with respect to appropriate treatment to be given to patients suffering from coagulopathies and to whom it may be necessary to administer anticoagulants, antifibrinolytic agents, thrombolytic agents, anti-platelet agents, or blood components in a quantity which must clearly be determined after taking into account the abnormal components or “factors” of the patient's blood and prior hemostasis treatment that may be contributing to the present hemostasis disorder.
Hemostasis is a dynamic, extremely complex process involving many interacting factors, which include coagulation and fibrinolytic proteins, activators, inhibitors and cellular elements, such as platelet cytoskeleton, platelet cytoplasmic granules and platelet cell surfaces. As a result, during activation, no factor remains static or works in isolation. The beginning of the coagulation process is platelet aggregation (FIG. 1a) and the initial phase of the enzymatic reaction. The end result of the coagulation process is a three dimensional network of polymerized fibrin(ogen) fibers which together with platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor bonding forms the final clot (FIG. 1b). A unique property of this network structure is that it behaves as a rigid elastic solid, capable of resisting deforming shear stress of the circulating blood. The strength of the final clot to resist deforming shear stress is determined by the structure and density of the fibrin fiber network and by the forces exerted by the participating platelets.
Thus, the clot that develops and adheres to the damaged vascular system as a result of activated coagulation and resists the deforming shear stress of the circulating blood is, in essence, a mechanical device, formed to provide a “temporary stopper,” which resists the shear force of circulating blood during vascular recovery. The kinetics, strength, and stability of the clot, that is, its physical property to resist the deforming shear force of the circulating blood, determine its capacity to do the work of hemostasis, which is to stop hemorrhage without permitting inappropriate thrombosis. This is exactly what the Thrombelastograph® (TEG®) hemostasis analysis system, described below, is designed to do, which is to measure the time it takes for initial fibrin formation, the time it takes for the clot to reach its maximum strength, the actual maximum strength, and the clot's stability.
Blood hemostasis analyzer instruments have been known since Professor Helmut Hartert developed such a device in Germany in the 1940's. One type of blood hemostasis analyzer is described in commonly assigned U.S. Pat. Nos. 5,223,227 and 6,225,126, the disclosures of which are hereby expressly incorporated herein by reference. This instrument, the TEG® hemostasis analysis system, monitors the elastic properties of blood as it is induced to clot under a low shear environment resembling sluggish venous blood flow. The patterns of changes in shear elasticity of the developing clot enable the determination of the kinetics of clot formation, as well as the strength and stability of the formed clot; in short, the mechanical properties of the developing clot. As described above, the kinetics, strength and stability of the clot provides information about the ability of the clot to perform “mechanical work,” i.e., resisting the deforming shear stress of the circulating blood; in essence, the clot is the elementary machine of hemostasis, and the TEGS® hemostasis analysis system measures the ability of the clot to perform mechanical work throughout its structural development. The TEG® hemostasis analysis system measures continuously all phases of patient hemostasis as a net product of whole blood components in a non isolated, or static fashion from the time of test initiation until initial fibrin formation, through clot rate strengthening and ultimately clot strength through fibrin platelet bonding via platelet GPIIb/IIIa receptors and clot lysis.
Heparin is one of the most widely prescribed anticoagulant drugs and has been very successful. However, heparin also has some potential adverse affects. As with any anti-coagulant, there is a risk of bleeding. Heparin has also been associated with an increased risk of osteoporosis, cutaneous reactions, and a condition referred to as heparin-induced thrombocytopenia (HiT).
HiT has been observed to occur in two forms. The first, type-I or non-immune HiT (HiT I), is commonly seen in patients receiving full dose intravenous unfractionated heparin. The fall in platelet count resulting from the introduction of heparin in HiT I is transient, is not associated with any adverse effects and is self-limiting insofar as it will resolve even if heparin therapy is continued. It is largely the result of heparin's binding directly to platelets.
Type-II, or immune-mediate HiT (HiT II), is the result of an antigen-antibody reaction. In HiT II, heparin-induced antibodies may form due to frequent exposure of patient blood to heparin. There is a high binding affinity between heparin and platelet factor four (PF4). Upon binding to the heparin molecule, PF4 exposes antigenic epitopes, which trigger the immune system and the production of immunoglobin G (IGH).
The IGH antibody binds to the antigen and to the platelets via the Fc fragment. Occupation of adjacent Fc receptors on the platelet membrane causes intense platelet activation resulting into lower platelet number (thrombocytopenia) and thrombosis in the form of white clot thrombi, leading to high risk of morbidity and mortality.
The Heparin-PF4-IGH referred to here as the HiT II complex.
There are two main classes of assays for laboratory diagnosis of HiT II: activation (functional) assays and antigen assays. The functional assays include the platelet aggregation assay and the serotonin release assay. The platelet aggregation assay is performed in the laboratory with a specificity>90%. The disadvantage is low sensitivity, <35%, i.e., a relatively high probability of false negative.
The serotonin release assay measures the release of serotonin from platelet aggregates. It relies on the aggregation of the platelets from the patient in the presence of heparin. This assay has high sensitivity and specificity. The disadvantage is that the assay is technically demanding and involves the use of radioactive materials. Of the various available functional assays available, platelet aggregation using washed platelets and platelet serotonin release are considered the most accurate.
The other class of assays is the antigen assays. The heparin-PF4 enzyme-linked immunosorbent assay (ELISA) relies on the specificity of the HiT IGH antibodies for the heparin-PF4 complex. This assay is 10 times more sensitive than the serotonin release assay for detecting heparin-induced antibodies. However, the heparin-PF4 ELISA is expensive and time consuming. The assay also responds to clinically insignificant antibodies more often than functional assays, and hence has a lower specificity, i.e., a relatively high probability of false positive.
Thus, most of the available laboratory tests for the diagnosis of HiT II are expensive, time-consuming, frequently contradictory and vary in sensitivity and specificity.
Because of the mortality and morbidity risk associated with treating a HiT II patient with additional heparin or platelets, the clinician must often resort, unnecessarily, to recommending another anticoagulant agent to be used instead of heparin when HiT II is suspected. However, other agents are more expensive and it is difficult or impossible to measure the extent of anticoagulation for proper dosing of the patient to prevent ischemic events. These anticoagulants also lack the agents necessary to reverse their anticoagulant effect, which may result with uncontrollable post-surgical hemorrhage.
Thus, there is a need for a method and apparatus for determining heparin-induced thrombocytopenia.